Provider: - Institution: - Data provided by Europeana Collections- Introduction: Melanoma, malignant neoplasm of melanocytes, despite of its immunogenicity is skin cancer with the highest mortality ...rate. In antitumor immunity im melanoma, natural killer (NK) cells as innate immune system effector cells play an important role as they are able to recognize malignantly transformed cells, lyse them by cytolitic enzymes (perforin and gramzymes) and also regulate adaptive and innate antitumor immunity by production of cytokines and chemokines. NK cells are present in many tissues and organs. In melanoma NK cells have been studied mostly in peripheral blood and in tumor tissue, while NK cells in regional lymph nodes (LN)s have not been investigated. NK cells are CD3-CD56+ and comprise two functionally distinct subsets, immunoregulatory, CD3-CD56bright+ and cytotoxic, CD3-CD56dim+. NK cell function is regulated by the balance of signals mediated by activating and inhibitory NK cell receptors. Activating receptors recognize stress induced ligands on tumor cells, while the inhibitory immunoglobulin-like killer receptors (KIR) by binding to MHC class I molecules inhibit NK cell function and enable self tolerance. NK cells are located mostly in T cell zone of LNs, where NK cell differentiation and maturation may occur. In physiological conditions, by peripheral circulation mostly immature CD3-CD56bright+ NK cell subset migrates to LNs and represents the majority of NK cell population in resting LNs. Endogenous cytokines in LN mediate maturation of this subset into cytotoxic CD3-CD56dim+ perforin+CD16+KIR+ subset that subsequently leaves LN. In pathological conditions, increase in CD16+KIR+ NK cell population in reactive LNs may be the consequence of increased production of innate immunity cytokines by dendritic cells, as well as Th1 cytokine production by antigen stimulated T cells in LNs, or selective migration of CD16+KIR+ NK cells into LNs. Among _c cytokines, IL-2 and IL-15 are most important for NK cell differentiation and maturation, and are also the most potent stimulators of NK cell cytotoxic activity. Objectives: As regional LNs represent first barrier to lymphogenic tumor invasion and considering the importance of microenvironment in shaping NK cell function and phenotype, the aim of this study was to compare functional and immunophenotypic characteristics of CD3-CD56+ NK cells from tumor-infiltrated (infiltrated) and not infiltrated regional LNs of melanoma patients. Since melanoma is an immunogenic tumor insensitive to irradiation and chemotherapy, the aim of this study was to investigate the ability of IL-2, cytokine that has been used more than two decades in metastatic melanoma treatment and IL-15, cytokine of novel generation which is essential for NK cell differentiation and maturation, to induce cytotoxic activity of NK cells from investigated regional LNs. Material and methods: Tissue samples of regional LNs were obtained from 45 melanoma patients in clinical stages II-IV immediately after surgical intervention. Mononuclear cells (MNC) were isolated from obtained LN samples. NK cell cytotoxic activity was evaluated against NK-cell sensitive K562 tumor cell line using radioactive 51Cr test on freshly isolated MNC as well as after 72 h and 7 day in vitro treatments in cell culture medium alone, medium supplemented with IL-2 (200 IU/ml) and with IL-15 (25 ng/ml) on 37ºC in humid atmosphere with 5% CO2. Flow cytometry method was used for evaluation of: IFNγ production in CD3- CD56+ NK cells, percentage of CD3-CD56+ NK cells, CD3-CD56dim+ and CD3- CD56bright+ NK cell subsets, as well as the expression of activating receptors (NKG2D, CD16), inhibitory KIRs (CD158a, CD158b) and activation antigens (CD25, CD69, HLA-DR) on CD3-CD56+ NK cells and their two subsets. After 72 h in vitro cytokine treatments perforin transcription was estimated by RT-PCR, while perforin, pSTAT1, pSTAT5 and Bcl-2 expression was estimated by Western blot. The investigated parameters between infiltrated and not infiltrated regional LNs were compared by statistical Mann Whitney test, while the significance of differences after cytokine in vitro treatments was evaluated by Wilcoxon rank sum test. For correlation analysis Spearman's correlation coefficient was evaluated. Results: In both not infiltrated and tumor- infiltrated regional LNs of melanoma patients were shown similar low levels of NK cell cytotoxic activity. Production of IFNγ, as another aspect of NK cell function, in entire CD3-CD56+ NK cell population was lower only in tumor-infiltrated LNs which could be influenced by immunosupresive cytokines and other biologically active molecules that might be of local or systemic origin. Tumor-infiltrated LNs also show higher percentage of CD3-CD56+ NK cells due to the increased presence of CD3-CD56dim+ subpopulation. This finding, together with increased percentage of CD16+ and CD158b+ CD3-CD56+ NK cells in tumor-infiltrated LNs, indicates more mature NK cell phenotype in LNs invaded by tumor cells. Furthermore, CD3-CD56+ NK cells in infiltrated LNs show increased expression of CD69 antigen which may indicate NK cell activation alongside with tumor invasion into LNs, while the expression of NKG2D cytototoxic receptor remained similar. The obtained differences in immunophenotypic parameters caused by tumor infiltration of LN are more apparent when more regional LNs are involved as well as if the degree of primary tumor invasion into skin was higher. After cytokine in vitro treatments it was shown that unlike IL-15, IL-2 treatment increased the percentage of CD3-CD56+ NK cell population by increasing CD3-CD56bright+ subset in both not infiltrated and tumor-infiltrated regional LNs of melanoma patients. Both IL-2 and IL-15 in vitro treatments increased low NK cell cytotoxicity, as well as perforin transcription and synthesis in both groups of regional LNs. These two cytokines on CD3-CD56+ NK cells in both regional LN groups increased the expression of early activating antigen CD69, cytotoxic NKG2D receptor, and also by increasing the expression of CD16 receptor and CD158b inhibitory KIR contributed to more mature NK cell phenotype. The effects of investigated cytokines are followed by increased level of phosphorylated STAT5 signaling molecule. Conclusion: In this study were analyzed the function and immunophenotype of NK cell population from regional LNs of malanoma patients which have not been investigated before. The obtained results indicate that metastatic invasion of melanoma into regional LNs did not alter cytotoxic function of resident NK cells, while it increased the percentage of CD3-CD56+ NK cells of more mature CD16+CD158b+ phenotype. In vitro treatments with IL-2, cytokine that has been used in melanoma treatment and IL-15, cytokine that is still under investigation for its therapeutic application, strongly induced the increase in NK cell cytotoxic activity which may indicate the therapeutic potential of this NK cell population in novel approaches such as adoptive NK cell transfer in melanoma patients.- Uvod: Melanom, maligni tumor melanocita, iako ima brojna imunogenična svojstva, predstavlja tumor kože sa najvišom incidencom smrtnosti. Za anititumorsku imunost u melanomu posebno su značajne ćelije prirodne ubice (NK ćelije), efektorske ćelije sistema urođene imunosti koje mogu neposredno da prepoznaju maligno transformisane ćelije i da ih liziraju citolitičkim enzimima (perforin i granzimi) kao i da produkcijom citokina i hemokina regulišu adaptivnu i urođenu amtitumorsku imunost. NK ćelije su prisutne u mnogim tkivima organima. Kod melanoma su uglavnom proučavane NK ćelije u perifernj krvi i u tumorskom tkivu, dok NK ćelije regionalnih limfnih čvorova (LČ) obolelih nisu do sada ispitivane. + NK ćelije su CD3 -CD56i obuhvataju dve funkcionalno različite subpopulacije, sjajno+ potmulo+ imunoregulatornu, CD3 -CD56i citotoksičnu, CD3 -CD56. Funkcija NK ćelija je regulisna balansom signala koji potiču od aktivacionih i inhibitornih NKćelijskih receptora. Aktivacioni receptori prepoznaju stresogene ligande na tumorskim ćelijama, dok inhibitorni ubilački receptori slični imunoglobulinima (engl. Immunoglobulin-like Killer Receptors, KIR) vezivanjem za MHC molekule klase I inhibiraju NK ćelijsku funkciju i pored toga omogućavaju toleranciju NK ćelija na ćelije sopstvenog organizma. NK ćelije su prisutne u T ćelijskoj zoni LČ, gde se odvija njihovo sazrevanje i diferencijacija. U fiziološkim uslovima u LČ perifernom cirkulacijom dospeva sjajno+ uglavnom nezrela (CD3 -CD56) NK ćelijska subpopulacija koja čini veći deo NK ćelijske populacije u mirujućim limfnim čvorovima. Pod uticajem endogenih potmulo+ citokina u LČ ova subpopulacija sazreva u citotoksičnu CD3 -CD56 +++ perforinCD16KIRsubpopulaciju koja zatim napušta LČ. U patološkim stanjima ++ dolazi do povećane zastupljenosti CD16KIRNK ćelija u reaktivnim LČ što može biti posledica povećane produkcije citokina urođene imunosti od strane dendritičnih ćelija, kao i Th1 citokina od strane stimulisanih T ćelija antigenima u LČ, kao i ++ selektivne migracije CD16KIRNK ćelija u LČ. Među citokinima γc familije, interleukini IL-2 i IL-15 su posebno značajni za diferencijaciju i sazrevanje NK ćelija, a takođe su pokazali i najizraženiji stimulativni efekat na citotoksičnu aktivnost NK ćelija. Ciljevi rada: S obzirom na to da regionalni LČ predstavljaju prvu barijeru u limfogenom širenju tumora kao i na značaj uticaja mikrosredine na funkciju i fenotip NK ćelija, cilj ovog rada bio je da se uporede funkcionalne i + imunofenotipske karakteristike CD3 -CD56NK ćelija u tumor-infiltrisanim (infiltirsanim) i neinfiltrisanim regionalnim LČ bolesnika sa melanomom. S obzirom na to da je melanom imunogeničan tumor koji je slabo osetljiv na zračnu i na hemioterapiju, cilj ovog rada bio je da se ispita sposobnost IL-2, citokina koji se već više od dve decenije primenjuje u lečenju metastatskog melanoma
